FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity

Genome editing technologies hold tremendous potential in biomedical research and drug development. Therefore, it is imperative to discover gene editing tools with superior cutting efficiency, good fidelity, and fewer genomic restrictions. Here, we report a CRISPR/Cas9 from Faecalibaculum rodentium, which is characterized by a simple PAM (5′-NNTA-3′) and a guide RNA length of 21–22 bp. We find that FrCas9 could achieve comparable efficiency and specificity to SpCas9. Interestingly, the PAM of FrCas9 presents a palindromic sequence, which greatly expands its targeting scope. Due to the PAM sequence, FrCas9 possesses double editing-windows for base editor and could directly target the TATA-box in eukaryotic promoters for TATA-box related diseases. Together, our results broaden the understanding of CRISPR/Cas-mediated genome engineering and establish FrCas9 as a safe and efficient platform for wide applications in research, biotechnology and therapeutics.


The authors should upload their formatted bioinformatics code to GitHub for the reviewers.
Otherwise, it is difficult to evaluate the identification pipeline.
Reviewer #2: Remarks to the Author: Cui et al. describe FrCas9, a previously uncharacterized Cas9 ortholog that recognizes a 5'-TA-3' PAM and that is active in genome editing in mammalian cells. In combination with other recent Cas9 editors that recognize different (CC, AA) two-nucleotide PAMs besides the GG of SpyCas9, this report provides a useful addition to the current roster of Cas9s. Importantly, they show that this platform can support base editing, where PAM availability (in a narrow window relative to the editing site) is crucial. This report could therefore be a valuable contribution to the field, though there are a number of problems with the draft that would need to be addressed first.
Although there are few addressable issues with the experiments (more on that below), in general the authors present a decent case that the PAM is indeed NNTA, that the activity in mammalian cells is strong, and that accuracy is sufficient for most editing purposes. However, the biggest problem by far is that they then take things too far and commit an unforced error, namely arguing unequivocally that this platform is superior to SpyCas9 in both activity and specificity. FrCas9 will be a very useful enzyme if it is as good, or even somewhat less good, than SpyCas9 in these respects, so (in my view) acceptance of this manuscript should not depend upon superiority over SpyCas9. The problem is that these claims of higher efficiency and accuracy would need to be backed by considerably more and better evidence than the authors provide. The numbers of guides and sites (with GUIDE-seq analyses) would need to be increased tremendously to get a reliable statistical sampling in support of these claims. The evidence would also need to go well beyond plasmid transfections in two transformed cell lines. We have no idea if any apparent activity/accuracy differences at any particular sites have to do with efficiency of transcription, translation, nuclear import, guide folding, guide loading, protein turnover, etc. etc. etc., rather than true efficiency differences. These other factors could exhibit cell-type-specific or cell-statespecific differences, and therefore preliminary indications of efficiency and accuracy may or may not pan out more broadly. Biochemical data would be crucial in support of the authors' claims, especially with respect to enzymatic efficiency, and there is essentially no biochemical analysis in this work. It would also be very helpful to know if the explicitly comparative experiments were done blind, as they should be. Overall, the manuscript would be better if the authors simply describe these preliminary indications of accuracy and efficiency as comparable to those of SpyCas9, pending deeper analyses at much larger numbers of sites.
Other concerns: 1. Lines 50-54: The authors invoke computational target site selections and protein engineering as the two strategies employed thus far to improve editing accuracy. They need to add guide engineering as a third, given previous reports about truncated guides (PMID 24463574), extended guides (24253446), and chemically modified guides (29377001, 29377001). 2. Lines 55-62: the claim that Cas9s other than SpyCas9s suffer from long PAMs was true once but is increasingly untenable. It is becoming a bit of a "straw man" argument and in this case is accompanied by cherry-picked examples and citations that omit recent Cas9s with two-nucleotide PAMs. 3. Line 80-81: "not close" should be defined more quantitatively and rigorously. 4. Lines 162-164: "directly compare" is misleading and not really true, because the guide sequences themselves are offset by one nucleotide. This is not as minor a change as it might sound because it could have substantial effects on guide folding and loading. 5. Lines 248-250: it makes no sense to invoke hydrogen bond stability in this context because PAMs are not unwound for recognition. How H-bond pairing by PAM residues could affect specificity is not explained, and such an attempt at explaining that claim would likely fail. 6. Lines 250-252: it is incorrect to state that longer guides necessarily imply or explain greater accuracy. See 1871108 (which is about ribozymes but uses logic that holds with other systems that use pairing for recognition) and 28125791.

Reviewer #1 (Remarks to the Author):
Major Concern 1. The authors utilize a depletion assay to assess PAM specificity.
However, from the text and figures, it is not clear about the details of the assay and whether it would faithfully recapitulate the PAM sequences of other known Cas9 effectors. In addition to providing more detailed text within the manuscript, I would ask the authors to conduct an additional PAM determination assay (PAMDA) that has recently been published and evaluate PAM sequences in a more relevant context: https://www.nature.com/articles/s41596-020-00465-2. I also ask that they conduct comparisons with a few other Cas9s of their choosing for validation.
Response: Thank you for the helpful comment. According to your advice, we purchased the p11-Cas9_random_PAM-site1 plasmid (Addgene #160132) and pCMV-T7-SpCas9-P2A-EGFP plasmid (Addgene #139987). Then, we constructed the pCMV-T7-FrCas9-P2A-EGFP plasmid and synthesized the in vitro transcription templates of SpCas9 and FrCas9 sgRNAs for PAM-site1. Next, we conducted the HT-PAMDA experiments with timepoints including 1 min, 8 min and 32 min. We generated high-throughput data with sequencing depth of ~15,000,000 reads per timepoint, assuring sufficient coverage to resolve up to 5 nt of the PAM preference.
To better visualize and understand the PAM preference from the HT-PAMDA data, we Major Concern 3a. The authors use GUIDE-Seq to evaluate on-and off-target efficiency. GUIDE-Seq is an internally controlled assay, not one that can be compared across samples. Thus, I would advise the authors to not use GUIDE-Seq to determine editing rate on target, but rather to calculate the ratio of on:off target reads as a measure of specificity at each site. Plotting these values across the different sites will provide a fuller understanding of FrCas9's specificity vs. SpCas9.  (8), SpCas9-HF1 (6), HiFi-Cas9 (12) and eSpCas9 (7) (Fig. 3f-g). As expected, the FrCas9 exhibited the highest on:off ratio in both sites (Fig. 3h). We have added the above results in Fig. 3f-h and revised our manuscript correspondingly (Page 9, Line 180-186). Major Concern 4. The authors present compelling data on TATA-box editing, which is the most promising editing route for FrCas9, due to its 5'-NNTA-3' PAM sequence.

Response
As the TATA box is upstream of the gene to be regulated, I suggest that the authors design sgRNAs upstream of the TATA box and demonstrate reduction of expression using CRISPRa rather than FrCas9 nuclease. A dFrCas9-VPR or -VP64 construct would be suffice. If FrCas9 CRISPRa activates expression better than SpCas9 CRISPRa, due to its optimal positioning at the TATA box, this would be a very compelling argument to use FrCas9 for CRISPRa/i screening.
Response: Thank you for the helpful comment. According to your advice, we tested FrCas9 CRISPRa using dFrCas9-VP64 directly targeting the TATA-box and compared its performance with dSpCas9-VP64 targeting the upstream of TATA-box.
These experiments were conducted in ABCA1, SOD1, GH1 and BLM2 genes in HEK293T cell line. The results showed that dFrCas9-VP64 enabled effective transcriptional activation. Moreover, the fold activation of dFrCas9-VP64 in ABCA1, GH1 and BLM2 was higher than that of dSpCas9-VP64, while the fold activation of SOD1 gene was comparable to that of dSpCas9-VP64 (Fig. 5g). Therefore, FrCas9 is a promising tool for CRISPR screening due to its unique 5'-NNTA-3' PAM. We  provides a useful addition to the current roster of Cas9s. Importantly, they show that this platform can support base editing, where PAM availability (in a narrow window relative to the editing site) is crucial. This report could therefore be a valuable contribution to the field, though there are a number of problems with the draft that would need to be addressed first.
Although there are few addressable issues with the experiments (more on that below), in general the authors present a decent case that the PAM is indeed NNTA, that the activity in mammalian cells is strong, and that accuracy is sufficient for most editing purposes. However, the biggest problem by far is that they then take things too far and commit an unforced error, namely arguing unequivocally that this platform is superior to SpyCas9 in both activity and specificity. FrCas9 will be a very useful enzyme if it is as good, or even somewhat less good, than SpyCas9 in these respects, so (in my view) acceptance of this manuscript should not depend upon superiority over SpyCas9. The problem is that these claims of higher efficiency and accuracy would need to be backed by considerably more and better evidence than the authors provide.
The numbers of guides and sites (with GUIDE-seq analyses) would need to be increased tremendously to get a reliable statistical sampling in support of these claims.
The evidence would also need to go well beyond plasmid transfections in two transformed cell lines. We have no idea if any apparent activity/accuracy differences at any particular sites have to do with efficiency of transcription, translation, nuclear import, guide folding, guide loading, protein turnover, etc. etc. etc., rather than true efficiency differences. These other factors could exhibit cell-type-specific or cell-state-specific differences, and therefore preliminary indications of efficiency and accuracy may or may not pan out more broadly. Biochemical data would be crucial in support of the authors' claims, especially with respect to enzymatic efficiency, and there is essentially no biochemical analysis in this work. It would also be very helpful to know if the explicitly comparative experiments were done blind, as they should be.
Overall, the manuscript would be better if the authors simply describe these preliminary indications of accuracy and efficiency as comparable to those of SpyCas9, pending deeper analyses at much larger numbers of sites.
Response: Thank you very much for the constructive suggestion. Indeed, we agreed that the data in our study was limited to draw such a claim. Therefore, we revised the manuscript according to your advice: Title: "A new CRISPR/Cas9 system with higher editing efficiency and fidelity compared to SpCas9" was changed into "A new CRISPR/Cas9 system with high editing efficiency and fidelity". Page 4, Line 69-70: "Further, we showed that FrCas9 achieved more efficient and safer genome editing than SpCas9 in human and HPV genome" was changed into "Further, we showed that FrCas9 could achieve comparable efficiency and specificity to SpCas9".
Page 10, Line 200-201: "FrCas9 exhibited superior efficiency and specificity than SpCas9 as an antiviral therapeutic tool" was changed into "FrCas9 exhibited remarkable efficiency and specificity as an antiviral therapeutic tool".
Therefore, FrCas9 may be more suited for genome engineering in mammalian cells than SpCas9 and will be a powerful and safe platform for biotechnological and clinical research" was changed into "FrCas9 hold high efficiency and specificity with simple 2-nuleotide PAM sequences (5'-NNTA-3'). Therefore, FrCas9 will be a powerful and safe platform for biotechnological and clinical research".
Page 12, Line 276-277: "Taken together, FrCas9 with unique PAM (5'-NNTA-3') sequences has superior efficiency and specificity than widely used SpCas9" was changed into "Taken together, FrCas9 with unique PAM (5'-NNTA-3') sequence has high efficiency and specificity". Response: Thank you for the helpful comment. As you suggested, we added the guide engineering as the third strategy in the introduction section. The manuscript was revised as below (Page 3, Line 52-54): "The third is to engineer guide RNA, including truncated gRNAs 2 , extended gRNAs 3 and chemically modified gRNAs 4 ".

Minor Concern 2.
Lines 55-62: the claim that Cas9s other than SpyCas9s suffer from long PAMs was true once but is increasingly untenable. It is becoming a bit of a "straw man" argument and in this case is accompanied by cherry-picked examples and citations that omit recent Cas9s with two-nucleotide PAMs.
Minor Concern 3. Line 80-81: "not close" should be defined more quantitatively and rigorously.
Response: Thank you for the constructive comment. Based on your suggestion, we revised the sentence into "The phylogenetic analysis shows that FrCas9 is dissimilar to SpCas9 at a distance of 1.80 ( Fig. 1a and  Minor Concern 4. Lines 162-164: "directly compare" is misleading and not really true, because the guide sequences themselves are offset by one nucleotide. This is not as minor a change as it might sound because it could have substantial effects on guide folding and loading. Response: Thank you for the helpful suggestion. We have changed "we could directly compare their genome editing efficiency and specificity in sequence with 5'-GGTA-3'." into "we compared their genome editing efficiency and specificity in sequence with 5'-GGTA-3'" (Page 8, Line 165-166).
Minor Concern 5. Lines 248-250: it makes no sense to invoke hydrogen bond stability in this context because PAMs are not unwound for recognition. How H-bond pairing by PAM residues could affect specificity is not explained, and such an attempt at explaining that claim would likely fail. Lines 250-252: it is incorrect to state that longer guides necessarily imply or explain greater accuracy. See 1871108 (which is about ribozymes but uses logic that holds with other systems that use pairing for recognition) and 28125791.
Response: Thank you for the kind comment. We agreed with your comment and deleted this context in our revised manuscript (Page 12, Line 257).